Whole-body optical molecular imaging of small animals is widely used to investigate fundamental biological processes in vivo and accelerate drug development. However, the actual technical implementations do not allow for robust quantitative rendering of 3D bio- distribution of the luminescent markers and require lengthy acquisition times incompatible with multiplexed studies. Our hypothesis is that by employing structured light illumination strategies and by using reconstruction algorithms based on the Monte Carlo method, the inverse optical molecular imaging problem can be solved accurately to provide quantitative spatial maps of molecular fluorescence markers distribution in the internal organs of small animals and simultaneously for multiply markers. The objective of this proposal is to develop, characterize and validate a novel pre-clinical imaging platform that will allow tracking in vivo multiple fluorophores in small animals. First, we intend to develop a fluorescence molecular small animal imager that will be able to provide quantitative fluorescent tomographic data for in vivo multiplexed study. Second, we propose to develop new reconstruction algorithm to perform multispectral time-resolved optical reconstructions. The algorithm will be based on the Monte Carlo method for accurate light propagation model in complex geometry and for complex illumination strategies. Third, we will characterize the performances of this new enabling platform to perform Fluorescence Resonance Energy Transfer (FRET) imaging in vivo. We will integrate a non-contact optical imager based on high-power NIR laser, Digital Micromirror Device (DMD) and time-resolved spectrophotometer. Reconstruction algorithms based on an adjoint Monte Carlo formulation will be developed concurrently to model light propagation in small animal and to perform fluorescent yield and lifetime reconstructions. After validating the imaging platform in vitro, whole-body in vivo small animal imaging of NIR-ligand labeled FRET pair will be performed. This new system will fill an important unmet need in pre-clinical studies. This research proposal is expected to revolutionize the investigation of fundamental biological processes in vivo and considerably accelerate drug development by providing a new imaging tool with high-sensitivity and high-throughput capability for molecular imaging studies in whole animals.